Innovative therapies based on autologous adipose-derived stem/stromal cells (ASC) are currently being evaluated for treatment of systemic sclerosis (SSc). Although paracrine angiogenic and antifibrotic effects are considered the predominant mechanisms of ASC therapeutic potential, the impact of SSc on ASC paracrine functions remains controversial. In this study, phenotype, senescence, differentiation potential, and molecular profile were determined in ASC from SSc patients (SSc-ASC) (n = 7) and healthy donors (HD-ASC) (n = 7). ASC were co-cultured in indirect models with dermal fibroblasts (DF) from SSc patients or endothelial cells to assess their pro-angiogenic and antifibrotic paracrine effects. The angiogenic activity of endothelial cells was measured in vitro using tube formation and spheroid assays. DF collagen and alpha smooth muscle actin (αSMA) content were quantified after five days of co-culture with ASC. Differentiation capacity, senescence, and mRNA profiles did not differ significantly between SSc-ASC and HD-ASC. SSc-ASC retained the ability to stimulate angiogenesis through paracrine mechanisms; however, functional assays revealed reduced potential compared to HD-ASC. DF fibrosis markers were significantly decreased after co-culture with SSc-ASC. Together, these results indicate that SSc effects do not significantly compromise the angiogenic and the antifibrotic paracrine properties of ASC, thereby supporting further development of ASC-based autologous therapies for SSc treatment.

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

(1) Background: The emergence of injectable “biologic” medication creates a new approach to treat osteoarthritis (OA). Among them, the use of intra-articular injection of PRP became widespread despite the absence of consensus regarding its optimal composition. The aim of this study was to retrospectively correlate an extensive biological characterization of injected PRP to the clinical responses of patients presenting knee OA. (2) Methods: This retrospective study included 75 patients with knee OA. Cartilage lesions were assessed using magnetic resonance imaging and the International Cartilage Regeneration Society (ICRS) classification. PRP extensive biological characterization was performed and patients’ subjective symptoms were recorded before injection and 3 and 6 months after injection using the Knee injury and Osteoarthritis Outcome Score (KOOS). Responders were defined by an improvement of 10 points on KOOS. (3) Results: At 6 months, 63.0% of the patients were responders. Impairment was characterized by a significantly higher proportion of patients with three compartments altered at baseline MRI and receiving a significantly higher dose of platelets compared to responders. (4) Conclusions: Single injection of pure PRP resulted in significant clinical improvement in the management of knee OA. Both baseline MRI and PRP biological features may be predictive factors of the clinical response, highlighting that a better understanding of action mechanism of PRP is still required.

Background : Even though the manufacturing processes of the stromal vascular fraction for clinical use are performed in compliance with the good-manufacturing practices applying to advanced therapy medicinal products, specifications related to stromal vascular fraction quality remain poorly defined. We analyzed stromal vascular fraction clinical batches from two independent good-manufacturing practices-compliant manufacturing facilities, the Swiss Stem Cell Foundation (SSCF) and Marseille University Hospitals (AP-HM), with the goal of defining appropriate and harmonized release acceptance criteria.Methods: This retrospective analysis reviewed the biological characteristics of 364 batches of clinical-grade stromal vascular fraction. Collected data included cell viability, recovery yield, cell subset distribution of stromal vascular fraction and microbiological quality. Results: stromal vascular fraction from SSCF cohort demonstrated a higher viability (89.33 % ± 4.30 %) and recovery yield (2.54 × 10⁵ ± 1.22 × 10⁵ viable nucleated cells (VNCs) per mL of adipose tissue) than stromal vascular fraction from AP-HM (84.20% ± 5.96% and 2.25 × 10⁵ ± 1.11 × 10⁵ VNCs). AP-HM batches were significantly less contaminated (95.71 % of sterile batches versus 74.15 %) The cell subset distribution was significantly different (higher proportion of endothelial cells and lower proportion of leukocytes and pericytes in SSCF cohort). Conclusions: Both centers agreed that a good-manufacturing practices-compliant stromal vascular fraction batch should exert a viability equal or superior to 80 %; a minimum recovery yield of 1.50 × 10⁵ VNCs per mL of adipose tissue; a proportion of adipose-derived stromal cells at least equal to 20 %; and a proportion of leukocytes under 50 %. In addition, a multiparameter gating strategy for stromal vascular fraction analyze is proposed.

La Fraction Vasculaire Stromale (FVS) est une suspension cellulaire hétérogène. L’activité synergique de l’ensemble des cellules la composant lui confère des propriétés biologiques multiple. L’apparente facilité de production de FVS a déclenché un engouement pour son utilisation en clinique. Pourtant de nombreux facteurs peuvent avoir une influence sur la qualité et la sécurité de ce MTI. Ce travail de thèse a pour objectif de caractériser l’impact de ces éléments technologiques et biologiques. Le premier volet de ce travail a consisté à développer un protocole de production standardisé, conforme aux Bonnes Pratiques de Fabrications, de la FVS permettant l’obtention d’une substance active optimisée. Une large étude rétrospective multicentrique de FVS de grade thérapeutique a été réalisée ensuite pour progresser vers la standardisation des contrôles qualités appliqués à la FVS. L’analyse de presque trois cents FVS cliniques a abouti à la proposition de critères libératoires. Le troisième volet de ce travail s’est intéressé à la caractérisation biologique de la FVS en contexte autologue de facteurs de risque vasculaire : l’âge et le diabète. L’impact de l’âge a été évalué grâce à un modèle de FVS issue de tissu adipeux périrénal de donneurs à critères élargis. Cette étude a mis en évidence une signature inflammatoire, avec une proportion de lymphocytes Natural Killer plus élevée au sein de la FVS et une expression de cytokines pro-inflammatoires, corrélée à l’âge. L’investigation de l’impact du diabète sur la FVS a montré l’existence d’un effet délétère marqué sur le contingent endothélial (augmentation de la sénescence et la réduction des propriétés angiogéniques).